Compositions and methods for manipulating carbon flux in cells

ABSTRACT

Nucleotide sequences and genetic constructs that can be used to regulate genes encoding enzymes that change carbon flux through metabolic pathways that lead to lactic acid or fumarate production in a host cell, such as a  R. oryzae  cell, are provided. Methods of manipulating carbon flux in a cell also are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/643,982, filed Jan. 14, 2005, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel nucleic acids and related methodsthat can be used to regulate genes encoding enzymes that manipulatecarbon flux through metabolic pathways.

BACKGROUND

Metabolic engineering of microorganisms is an effective means to producecommercially a number of chemicals useful for a variety of applications,including production of polymer monomers and food additives (see, e.g.,Lee, S. Y., et al. Macromol. Biosci. 4:157-164 (2004)).

As an example, fumaric acid is an organic acid widely found in nature.In humans and other mammals, fumaric acid is a key intermediate in thetricarboxylic acid cycle for organic acid biosynthesis (also known asthe Krebs cycle or the citric acid cycle). Fumaric acid is also anessential ingredient in plant life. Fumaric acid is the strongestorganic food acid in titratable acidity and in sourness. In one example,commercial fumaric acid is made from N-butane that is oxidized to maleicacid that is then isomerized to fumaric acid. Production of fumaric acidby bioprocess methods has potential to avoid synthetic productionprocesses that often are more costly than bioprocess methods.

As an additional example, lactic acid (lactate) is used in the foodindustry as an additive for preservation, flavor, and acidity. It isalso used for the manufacture of poly-lactic acid, a biodegradableplastic, and ethyl lactate, an environmentally friendly nonchlorinatedsolvent. Worldwide, in excess of 100,000 tons of lactic acid is producedannually, with predictions of an increasing demand. The growth in demandis attributable to the poly-lactic acid and ethyl acetate products.

In a number of microorganisms, lactic acid is normally produced frompyruvic acid (pyruvate). The reaction also occurs in the cells of higherorganisms when oxygen is limited. Glycolysis is the sequence ofreactions that converts glucose into pyruvic acid (pyruvate). Glycolysiscan be carried out anaerobically. Pyruvic acid has a number of fatesdepending on where the chemical reaction takes place and whether thereaction takes place in the presence or absence of oxygen.

As shown in FIG. 1, under aerobic conditions, pyruvic acid can beconverted to acetyl-CoA by pyruvate dehydrogenase. Under anaerobicconditions, pyruvic acid can be converted to ethanol (alcoholicfermentation) or lactic acid (e.g., in contracting muscle). Theconversion of pyruvic acid to lactic acid is catalyzed by lactatedehydrogenase (LDH). The efficiency of lactic acid fermentation can bequantified as the percent yield of lactate from glucose or as a decreasein the levels of co-products (e.g., glycerol, ethanol, and fumarate)found in the fermentation broth.

Lactic acid is often manufactured using Lactobacilli, which typicallyhas specialized growth requirements and is unable to produce significantamounts of lactic acid below pH 4. (Skory, C. D. J. Ind. Microbiol.Biotechnol. 30:22-27 (2003)). Alternatively, maintenance of neutral pHresults in decreased product solubility in the form of salts andrequires further processing to regenerate the acid from the resultinglactate salt.

Saccharomyces cerevisiae is a hearty, acid-tolerant microorganism thatis amenable to industrial processes. In these microorganisms, however,the major product of pyruvate metabolism is ethanol, by way of pyruvatedecarboxylase. Skory reported the production of lactic acid in a yeast,S. cerevisiae, expressing an ldh gene derived from Rhizopus oryzae. (J.Ind. Microbiol. Biotechnol. 30:22-27, (2003)). Skory demonstrated anincrease in lactic acid production in the recombinant yeast.Nevertheless, despite the increase in lactic acid production, themajority of carbon was diverted into ethanol. In the same report, whenlactic acid production was studied in a S. cerevisiae mutant straindeficient in ethanol production, diminished ethanol production wasobserved, but the efficiency of lactic acid production also decreased.

Anderson et al. demonstrated that ldh activity had little or no effecton the flux of carbon to lactic acid in Lactococcus lactis. Eur. J.Biochem., 268:6379-6389 (2001). Despite increasing the expression andactivity of ldh to beyond that found in wild-type L. lactis, researchersobserved no change in the flux of carbon to lactic acid.

Lactic acid can be synthesized chemically, but such synthesis results ina mixture of D and L isomers. The products of microbiologicalfermentation depend on the organism used and also may include a mixtureof the two isomers or individual isomers in a stereospecific form. Thedesired stereospecificity of the product depends on the intended use;however, L-(+)-lactic acid is the form desired for most applications(Skory, C. D. Appl. Environ. Microbiol. 66:2343-2348 (2000)).

U.S. Pat. No. 6,528,636 describes R. oryzae (ATCC 9363) as a lactic acidproducer found in the Rhizopus genus. Rhizopus is a filamentous fungusthat is commercially versatile and used in the production of fermentedfoods, industrial enzymes such as glucoamylase and lipase,corticosteroids, chemicals such as glycerol and ethanol, as well asorganic acids such as lactic acid and fumaric acid.

Production levels of different metabolites vary tremendously among theRhizopus species, with some species producing predominantly lactic acidand others producing primarily fumaric acid. An ideal lacticacid-producing Rhizopus strain would produce little or none of thesemetabolites, since their production depletes sugars that could be usedfor conversion to lactic acid.

Ethanol is believed to be produced by most Rhizopus species primarily inlow oxygen conditions. While Rhizopus is not typically considered anorganism that grows under anaerobic conditions, it does possess ethanolfermentative enzymes that allow the fungus to grow for short periods inthe absence of oxygen.

U.S. Pat. No. 4,877,731 discusses that fumaric acid production has beenwell studied in Rhizopus and that the fumarase gene also has beenisolated. Synthesis of fumarate is believed to occur primarily throughthe conversion of pyruvate to oxaloacetate by pyruvate carboxylase.Conditions leading to increased fumaric acid usually are associated withaerobic growth in high glucose levels and low available nitrogen.Accumulation of fumarate often is a problem with lactic acid production,because its low solubility can lead to detrimental precipitations thatcompromise fermentative efficiency.

Glycerol is also a by-product that often is produced by Rhizopus grownin high glucose-containing medium. Glycerol is thought to accumulate inRhizopus in a manner similar to that found in Saccharomyces (U.S. Pat.No. 6,268,189).

Oxaloacetate is also produced by Rhizopus. Pyruvate carboxylase [EC6.4.1.1] is a member of the family of biotin-dependent carboxylaseswhich catalyzes the carboxylation of pyruvate to form oxaloacetate withconcomitant ATP cleavage. The resulting oxaloacetate can be used for thesynthesis of glucose, fat, and some amino acids or other derivatives.The enzyme is highly conserved and is found in a wide variety ofprokaryotes and eukaryotes. During fermentation by Rhizopus oryzae,pyruvate is primarily converted to lactic acid, but other by-productssuch as fumaric acid, ethanol and glycerol are also produced. In thisfungus, there is evidence that fumaric acid production is predominantlyfrom cytosolic oxaloacetate that is converted from pyruvate by pyruvatecarboxylase (Osmani, S. A., et al., Eur. J. Biochem. 147:119-128(1985)).

Active pyruvate carboxylase consists of four identical subunits arrangedin a tetrahedron-like structure. Each subunit contains three functionaldomains: the biotin carboxylation domain, the transcarboxylation domainand the biotin carboxyl carrier domain (Jitrapakdee, S., et al.,Biochem. J. 340:1-16 (1999)). Pyruvate carboxylases contain theprosthetic group biotin, which is covalently bound to the amino group ofa specific lysine residue. The overall reaction catalyzed by pyruvatecarboxylase involves two partial reactions that occur at spatiallyseparate subsites within the active site, with the covalently boundbiotin acting as a mobile carboxyl group carrier. In the first partialreaction, biotin is carboxylated using ATP and HCO₃ ⁻ as substrates,while in the second partial reaction, the carboxyl group fromcarboxybiotin is transferred to pyruvate (Attwood, P. V., Int. J.Biochem. Cell Biol. 27:231-249 (1995)).

Pyruvate carboxylase was first described by (Utter, M. F., et al., J.Biol. Chem. 235:17-18 (1960)) in the course of defining thegluconeogenic pathway in chicken liver. Native pyruvate carboxylase froma number of sources, including bacteria, yeast, insects and mammals,consists of four identical subunits of approximately 120-130 kDa.Pyruvate carboxylases from many sources possess a reactive lysineresidue that is essential for full enzymatic activity. Sequencing ofcDNA encoding pyruvate carboxylase, as well as limited proteolysis andprimary structure comparisons, have shown that pyruvate carboxylasesfrom different species contain ATP, pyruvate, and biotin binding domains(Jitrapakdee and Wallace (1999); Koffas, M. A., et al., Appl. Microbiol.Biotechnol. 50:346-352 (1998)). In S. cerevisiae there are two pyruvatecarboxylase isoenzymes (PYC1 and PYC2) encoded by separate genes(Stucka, R., et al., Mol. Gen. Genet. 229:307-315 (1991); Walker, M. E.,et al., Biochem. Biophys. Res. Commun. 176:1210-1217 (1991)) while inmammals, no tissue-specific isoenzymes have been reported. Pyruvatecarboxylase is most effectively activated by long-chain acyl-CoAderivatives, such as palmitoyl-CoA, and is inhibited by aspartate and2-oxoglutarate (Osmani, S. A., et al., Ann. N.Y. Acad. Sci. 447:56-71(1985)).

Fermentations with the fungus Rhizopus are often advantageous becausethe organism is able to produce optically metabolites, such as pureL-(+)-lactic acid. Therefore, the quality of the final product isconsidered to be superior to that obtained by bacterial fermentations.Furthermore, L-(+)-lactic acid is more desirable for making poly-lacticacid. (U.S. Pat. No. 6,268,189). Additionally, Rhizopus can grow inchemically simple medium without the need for complex components such asyeast extracts (Skory, C. D. Curr. Microbiol. 47:59-64 (2003)).Nevertheless, the efficiency of lactic acid and fumaric acid production(the amount of available carbon diverted to lactate or fumarateproduction) in Rhizopus generally is low as compared to bacterialfermentations. There also is little known in the art about generegulatory elements for Rhizopus. There is a need for a method ofincreasing the efficiency and amount of lactate and fumarate productionin Rhizopus.

SUMMARY

Provided herein are genes and genetic elements useful in modifying hostcells, such as, without limitation, microorganisms. Further, the methodsand compositions of the invention are useful for overexpressing, forexample, and without limitation, specific metabolites in themicroorganism, such as, without limitation, fumaric acid, lactic acid,and glycerol. Methods of manipulating carbon flux in a microorganismsuch as R. oryzae also are provided.

In one embodiment, an isolated polynucleotide is provided comprising apromoter such as a Rhizopus transcription elongation factor (tef) genepromoter or, in another embodiment, Rhizopus ribosomal RNA cluster (rRNAcluster) gene promoter. In one embodiment, the isolated polynucleotidecomprises a promoter such as a Rhizopus oryzae transcription elongationfactor (tef) gene promoter contained within a sequence shown in one ofFIGS. 2, 3 and SEQ ID NO:1 and SEQ ID NO:2 as well as a Rhizopus oryzaeribosomal RNA cluster (rRNA cluster) gene promoter contained within asequence shown in FIG. 10, SEQ ID NO:10 and SEQ ID NO:11. The isolatedpolynucleotide can comprise an expressed sequence, such as an openreading frame or a sequence encoding an antisense RNA or an interferingRNA operably linked to the promoter. In other embodiments, the expressedsequence encodes one of an siRNA and an antisense RNA directed to one ofpyruvate dehydrogenase and pyruvate decarboxylase. In certainembodiments, the open reading frame encodes, for example, lactatedehydrogenase, pyruvate carboxylase, and phosphoenolpyruvatecarboxylase. The polynucleotide may be contained within a vector and/ora host cell.

Also provided is the sequence of a novel pyruvate carboxylase gene (SEQ.ID NO:6) and a protein product encodedthereof (SEQ ID NO:8) obtainedfrom R. oryzae.

In another embodiment, a method is provided for manipulating carbon fluxin a microorganism comprising: culturing a cell containing apolynucleotide capable of expressing a sequence for manipulating carbonflux in a cell (for example, a sequence as described supra) andrecovering one of lactic acid, glycerol and fumaric acid from theculture medium.

In another embodiment, a selectable marker for more efficient metabolicengineering of Rhizopus is provided.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asdescribed and claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of common metabolic pathways in R. oryzae, with aPEP carboxylase pathway introduced by expression of phosphoenolpyruvatecarboxylase gene (pepc) shown by the dotted line.

FIG. 2 shows the full length sequence of the tef gene promoter isolatedfrom R. oryzae. (SEQ ID NO:1). The TATA box and ATG start codon areshown underlined.

FIG. 3 shows a truncated sequence of the tef gene promoter isolated fromR. oryzae. (SEQ ID NO:2). The TATA box and ATG start codon are shownunderlined.

FIG. 4 shows a portion of the external transcribed spacer (ETS) regionof the 18s subunit of the ribosome isolated from R. oryzae. (SEQ IDNO:3).

FIG. 5 shows a comparison of nucleotide sequences pyruvate dehydrogenasefrom the genomic sequence “g” and the expressed sequence “c” of R.oryzae (SEQ ID NO:4 and SEQ ID NO:5, respectively).

FIG. 6 shows a cDNA sequence (SEQ ID NO:6), genomic DNA sequence (SEQ IDNO:7), and a protein sequence (SEQ ID NO:8) of R. oryzae pyruvatecarboxylase. The open reading frame encodes a protein of 1178 aminoacids. The intron, 61 bp, is typed in italic lowercase.

FIG. 7 shows the cDNA and protein sequence of Medicago sativaphosphoenolpyruvate carboxylase (SEQ ID NO:9).

FIG. 8 shows conserved domains among R. oryzae, S. cerevisiae, A. niger,A. terreus, P. pastoris, and S. pombe pyruvate carboxylase proteins. Thetwo ATP binding domains (amino acids 187-193 and 311-318 of the proteinsequence provided in FIG. 6, underlined) and the biotin binding domain(amino acids 1138-1141 of the protein sequence provided in FIG. 6,underlined) are 100% conserved, while the pyruvate binding domain (aminoacids 603-625 of the protein sequence provided in FIG. 6, underlined,with W₆₂₂ being the putative pyruvate binding site) is 89% conservedamong these fungal proteins.

FIG. 9 is a Southern blot of total genomic DNA from R. oryzae digestedwith restriction enzymes PstI, BamHI, or EcoRI showing relative copynumbers of the pyruvate carboxylase (pyrC) containing plasmid.

FIG. 10 shows a full length sequence of nucleotides 1-1043 of the rRNAcluster gene promoter region isolated from R. oryzae (SEQ ID NO:10). TherRNA cluster core promoter is shown in italics (SEQ ID NO:11).

DETAILED DESCRIPTION

Provided herein are methods and compositions of matter useful in themanipulation of carbon flux in microorganisms, typically in members ofthe Rhizopus genus, and most typically in R. oryzae. As a non-limitingexample, the manipulation of R. oryzae metabolic pathways depicted inFIG. 1 is facilitated by the methods and compositions of matterdescribed herein. Tools for manipulating carbon flux described hereininclude novel promoters and/or gene sequences, as well as portionsthereof and sequences complementary thereto which can be used inantisense and siRNA methods.

It is to be understood that certain descriptions of the presentinvention have been simplified to illustrate only those elements andlimitations that are relevant to a clear understanding of the presentinvention, while eliminating, for purposes of clarity, other elements.Those of ordinary skill in the art, upon considering the presentdescription of the invention, will recognize that other elements and/orlimitations may be desirable in order to implement the presentinvention. However, because such other elements and/or limitations maybe readily ascertained by one of ordinary skill upon considering thepresent description of the invention, and are not necessary for acomplete understanding of the present invention, a discussion of suchelements and limitations is not provided herein. As such, it is to beunderstood that the description set forth herein is merely exemplary tothe present invention and is not intended to limit the scope of theclaims.

Other than in the examples herein, or unless otherwise expresslyspecified, all of the numerical ranges, amounts, values and percentages,such as those for amounts of materials, elemental contents, times andtemperatures of reaction, ratios of amounts, and others, in thefollowing portion of the specification and attached claims may be readas if prefaced by the word “about”, even though the term “about” may notexpressly appear with the value, amount, or range. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains errornecessarily resulting from the standard deviation found in itsunderlying respective testing measurements. Furthermore, when numericalranges are set forth herein, these ranges are inclusive of the recitedrange end points (end points may be used). When percentages by weightare used herein, the numerical values reported are relative to the totalmass weight. Those of skill in the art recognize that percent massweight and actual mass weight are interconvertable.

All referenced patents, patent applications, publications, sequencelistings, electronic copies of sequence listings, or other disclosurematerial are incorporated by reference in whole but only to the extentthat the incorporated material does not conflict with existingdefinitions, statements, or other disclosure material set forth in thisdisclosure. As such, and to the extent necessary, the disclosure asexplicitly set forth herein supersedes any conflicting materialincorporated herein by reference. Any material, or portion thereof, thatis said to be incorporated by reference herein, but which conflicts withexisting definitions, statements, or other disclosure material set forthherein will only be incorporated to the extent that no conflict arisesbetween that incorporated material and the existing disclosure material.The articles “a,” “an,” and “the” are used herein to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one or more elements, andthus, possibly, more than one element is contemplated, and may beemployed or used.

As used herein, the term “auxotroph” refers to an organism that requiresa specific growth factor (for example, an amino acid or sugar) for itsgrowth. A “bradytroph” refers to an organism that does not necessarilyrequire a specific growth factor for its growth, but which produces acertain growth factor in lower amounts than a wild-type (w.t.) organism.

As used herein, the term “fumaric acid” refers to trans1,2-ethylenedicarboxylic acid in either the free acid or salt form. Thesalt form of fumaric acid is referred to as “fumarate” regardless of theanion, for example and without limitation, carbonate (e.g., neutralizingvia calcium carbonate) or hydroxide (e.g., neutralizing via ammoniumhydroxide).

By the term “lactic acid” is meant 2-hydroxypropionic acid in either thefree acid or salt form. The salt form of lactic acid is referred to as“lactate” regardless of the anion, for example and without limitation,carbonate (e.g., neutralizing via calcium carbonate) or hydroxide (e.g.,neutralizing via ammonium hydroxide).

By the term “gene” is meant a segment of nucleic acid, DNA or RNA, whichencodes and is capable of expressing a specific gene product. A geneoften produces a protein or polypeptide as its gene product, but in itsbroader sense, a gene can produce any desired product, whether theproduct is a protein, polypeptide or nucleic acid. Functional orstructural nucleic acid, such as, without limitation, rRNA, ribozymes,antisense RNA or interfering RNA (e.g., siRNA) also may be considered“gene products.”

A “gene” contains an “expressed sequence” that can encode not only aprotein or polypeptide, but a structural or functional nucleic acid,such as an antisense or siRNA. A gene may also contain sequencescontaining regulatory elements, such as, without limitation, promoters,enhancers and terminators; such regulatory elements may be “operablylinked,” most typically in an appropriate proximity to each other. Suchpromoters operate in cis (attached to each other on the same nucleicacid molecule) to cause expression of “a gene product.” The choice ofgene constituents, such as the particular combination of regulatoryelements and expressed sequence, will dictate the conditions ofexpression. For example, a constitutive promoter, such as the CMV(cytomegalovirus) promoter, coupled to an expressed sequence will causeconstitutive expression of the expressed sequence when transferred intoa suitable host cell. A promoter is considered constitutive if itfunctions to promote transcription of a gene under normal growthconditions. A constitutive promoter is not tissue specific ordevelopmentally specific, has broad cross-species tropism, and typicallydoes not vary substantially in its expression under normal growthconditions.

A “gene” can include introns or other DNA sequences that can be splicedfrom the final RNA transcript. An expressed DNA sequence that encodes aprotein or peptide (“protein encoding sequence”) includes an openreading frame (ORF). The protein encoding sequence may compriseintervening introns. Further, the term “gene” includes expressedsequences as well as non-expressed sequences. All DNA sequences providedherein are understood to include complementary strands unless otherwisenoted. Furthermore, RNA sequences can be prepared from DNA sequences bysubstituting uracil for thymine, and are included in the scope of thisdefinition and the invention, along with RNA copies of the DNA sequencesof the invention isolated from cells.

By the term “oligonucleotide” is meant a nucleic acid of from about 7 toabout 50 bases though they are more typically from about 15 to about 35bases. Oligonucleotides are useful as probes or primers for use inhybridization or amplification assays such as Southern or Northernblots; molecular beacon; polymerase chain reaction (PCR); reversetranscriptive PCR (RT-PCR); quantitative RT-PCR (QRT-PCT), e.g., TAQMAN;isothermal amplification methods, such as NASBA (nucleic acidsequence-based amplification); and rolling circle amplification,including use of padlock probes. The oligonucleotides of the inventioncan be modified by the addition of peptides, labels (includingfluorescent, quantum dot, or enzyme tags), and other chemical moietiesand are understood to be included in the scope of this definition andthe invention.

As used herein, in the context of the novel nucleotide sequencesdescribed herein, a nucleic acid is “specific to” a given sequence, suchas the pyruvate carboxylase cDNA and genomic sequences provided, if itcan hybridize specifically to a given sequence under stringentconditions, such as, without limitation, 0.2×SSC at 65° C. or in a PCRreaction under typical reaction (annealing) temperatures. Typically, onesequence is “specific” to a reference sequence if the nucleic acid has90 to 100% homology (sequence identity) to the reference sequence.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”. Asused herein, “reference sequence” is a defined sequence used as a basisfor sequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be 30, 40, 50, 100,or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches. Methods of alignment of sequencesfor comparison are well known in the art. Thus, the determination ofpercent sequence identity between any two sequences can be accomplishedusing a mathematical algorithm. Non-limiting examples of suchmathematical algorithms are the algorithm of Myers and Miller (1988)CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981)Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignmentmethod of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448;the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,and PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used. Seethe National Center for Biotechnology Information website on the worldwide web at ncbi.nlm.nih.gov. Alignment may also be performed manuallyby inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3 and thenwsgapdna.cmp scoring matrix; or any equivalent program thereof. By“equivalent program” is intended to mean any sequence comparison programthat, for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the GCG Wisconsin Genetics Software Package for protein sequences are8 and 2, respectively. For nucleotide sequences the default gap creationpenalty is 50 while the default gap extension penalty is 3. The gapcreation and gap extension penalties can be expressed as an integerselected from the group of integers consisting of from 0 to 200. Thus,for example, the gap creation and gap extension penalties can be 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins, it is recognizedthat residue positions which are not identical often differ byconservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g., charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. When sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, preferably at least 80%, more preferably at least 90%, andmost preferably at least 95%, compared to a reference sequence using oneof the alignment programs described using standard parameters.

In the context of the sequences provided herein, a sequence is specificto that reference sequence if, under any given reaction condition thatcan be used to distinguish one sequence from another, such as, withoutlimitation, PCR, Southern blot or Northern blot, the nucleic acid canhybridize specifically to a nucleic sequence provided herein, but not toother sequences, such as sequences from other species including withoutlimitation those of S. cerevisiae, A. niger, A. terreus, P. pastoris,and S. pombe. Thus, in a nucleic acid detection assay, a probe/primer is“specific to” a sequence if it can bind to a specific transcript ordesired family of transcripts extracted from a specimen, to thepractical exclusion (i.e., does not interfere substantially with thedetection assay) of other sequences. In a PCR assay, primers arespecific to a reference sequence if they specifically amplify a portionof that sequence, to the practical exclusion of other sequences in asample.

As used herein, a “primer” or “probe” for detecting a specific nucleicacid species is any primer, primer set, and/or probe that can beutilized to detect and/or quantify the specific nucleic acid species. A“nucleic acid species” can be a single nucleic acid species,corresponding to a single gene, or can be nucleic acids that aredetected by a single common primer and/or probe combination.

By the term “host cell” is meant any prokaryotic or eukaryotic cellwhere a desired nucleic acid sequence has been introduced into the cell.The metabolic processes and pathways of such a host cell are capable ofmaintaining, replicating, and/or expressing a vector containing aforeign gene or DNA molecule. There are a variety of suitable hostcells, including but not limited to bacterial, fungal, insect,mammalian, and plant cells, that can be utilized in various ways (forexample, as a carrier to maintain a plasmid comprising a desiredsequence). Representative microbial host cells include, but are notlimited to, fungal cells such as Rhizopus ssp., Saccharomyces ssp.,Streptomyces ssp., Pichia ssp., Aspergillus ssp., and bacterial cellssuch as Lactobacillus ssp., Escherichia ssp., Corynebacterium ssp.,Brevibacterium ssp., Pseudomonas ssp., Proteus ssp., Enterobacter ssp.,Citrobacter ssp., Erwinia ssp., Xanthomonas ssp., Flavobacterium ssp.,Streptococcus ssp., Lactococcus ssp., Leuconostoc ssp., and Enterococcusssp. In one embodiment, the host cell is Rhizopus oryzae. In anotherembodiment, the host cell is Escherichia coli.

By the term “polynucleotide” is meant any single-stranded sequence ofnucleotide, connected by phosphodiester linkages, or any double-strandedsequences comprising two such complementary single-stranded sequencesheld together by hydrogen bonds. Unless otherwise indicated, eachpolynucleotide sequence set forth herein is presented as a sequence ofdeoxyribonucleotides (abbreviated A, G, C and T). The term“polynucleotide” encompasses DNA molecules or polynucleotide, sequencesof deoxyribonucleotides, and RNA molecules or polyribonucleotides andcombinations thereof

By the term “promoter” is meant a DNA sequence within a larger DNAsequence that provides or defines a site to which RNA polymerase canbind and initiate transcription. The promoters described herein can beused to over-express or up-regulate, for example, and withoutlimitation, genes encoding enzymes that increase carbon flux to lacticacid, fumarate, and other desired metabolites during changes infermentation conditions.

By the term “carbon flux” is meant the biochemical pathway by whichcarbon is metabolized in an organism. A change in carbon flux,therefore, is a change in the metabolic processing of carbon in responseto a change in the organism or its environment. Carbon flux may bechanged in any manner, including but not limited to changing theenvironment of the organism, such as limiting oxygen and/or changing theexpression of genes and gene products in the organism (e.g. introducingheterdogous gene sequences).

An “equivalent” of a given reference nucleotide sequence or elementcontained therein is a nucleotide sequence containing, as compared tothe reference nucleotide sequence, all elements of that referencenucleotide sequence, such that the characteristic function of thatreference nucleic acid or peptide is retained. Those of skill in the artunderstand that a functional protein may be encoded by equivalent DNAsequences due to degeneracy in the genetic code. For example, one codonmay be substituted for another, yet encode the same amino acid, such as,for example and without limitation, in reference to the Ala codon, thesubstitution of GCC or GCG for GCA. In the case of proteins, a sequencecan contain amino acids that represent conservative amino acidsubstitutions, including but not limited to, the conservativesubstitution groups: Ser and Thr; Leu, Ile and Val; Glu and Asp; and Glnand Asn. A sequence as claimed herein thus includes the referencedsequence as well as its equivalents due to degeneracy in the geneticcode. Conservative substitutions also can be determined by othermethods, such as, without limitation, those used by the BLAST (BasicLocal Alignment Search Tool) algorithm, the BLOSUM Substitution ScoringMatrix, and the BLOSUM 62 matrix (see also, for example, Altschul etal., Methods in Enzymology 266:460-479 (1996)). Importantly,“equivalents” and “conserved equivalents” of a reference nucleic acid orpeptide/protein substantially retain or enhance the function of thereference nucleic acid or peptide/protein.

As used herein, a “tef promoter” or “tef Pol II promoter” is thepromoter for transcription of translation elongation factor. See, forexample, FIGS. 2 and 3; and SEQ ID NO:1 and SEQ ID NO:2. Likewise, an“rRNA cluster promoter ” is the promoter for transcription of ribosomalRNA such as the 5s (comprising the NTS1 promoter region) and 18s(comprising the NTS2 region) ribosomal RNA. Those of skill in the artrecognize that ribosomal DNA (rDNA) in eukaryotes is arranged intandemly repeated units containing the coding regions for 18S, 5.8S, and28S ribosomal RNA separated by spacers. A large intergenic spacer (IGS)separates the 28S and 18S coding regions, and contains signals fortranscription initiation and termination. The structure of the 35Spre-mRNA cluster is:NTS1::5S::NTS2::5′ETS::18S::ITS1::5.8S::ITS2::28S::3′ETS. The internaltranscribed spacers (ITS), which separate the 5.8S gene from the 18S and28S genes on either side of it, contain motifs responsible for thecorrect splicing of the mature 18S, 28S and 5.8S rRNA molecules from theprimary rRNA transcript wherein the promoter regions drive expression ofsuch rRNA. Examples of an rRNA cluster promoter sequence include thatshown in FIG. 10 and the sequence listed in SEQ ID NO:10 and SEQ IDNO:11.

In the context of the promoters described herein, equivalents of thosepromoters substantially retain the promoter activity, host cell tropismand strength of the promoter. Methods of making “equivalent” promotersinclude any of the large variety of genetic engineering and/ormutational methods known to those of skill in the art. These methods canbe used to create nucleic acid substitutions, deletions or insertionsthat do not substantially affect the promoter function. For example, andwithout limitation, in the case of the tef promoter (see, for example,FIGS. 2 and 3; and SEQ ID NO:1 and SEQ ID NO:2), in the region locatedbetween the TATA box and the downstream transcription start site (AUG),one or more nucleotides may be inserted, deleted or substituted withoutsubstantially decreasing promoter function. Similarly, other cis-actingelements present in the tef promoter, such as those found 5′ to the TATAbox (bases 735 to 739 of SEQ ID NO:1, with the ATG start codon at bases777 to 779; bases 208 to 213 of SEQ ID NO:2, with the ATG start codon atbases 251 to 253), may be retained, yet one or more nucleotides betweenthose cis-acting elements may be inserted, deleted or modified withoutsubstantially decreasing promoter function. Even small 1 or 2 nucleotidesubstitutions, insertions and deletions within promoter elements may betolerated without substantial loss of promoter function. As such,“equivalents” of the tef promoter contain sequences having at leastabout 90%, preferably at least about 95% and most preferably at leastabout 97.5% sequence identity with the sequences of the invention. Bothsequences presented in SEQ ID NO:1 and 2 retain the essential promotercharacteristics of the tef promoter.

As with the tef promoter, certain portions of the rRNA cluster promoterare necessarily substantially conserved in “equivalents,” while othersare not. As discussed herein, and as is well-known in the art, Pol Ipromoters such as the rRNA cluster promoters contain a core element andan upstream control element (“UCE”). As such, nucleotide sequencesbetween those elements need not be conserved, only their generalspacing. Thus, outside the core and UCE sequences, any nucleotide can bedeleted, inserted or substituted, so long as the ability of the promoterto cause expression of an operably linked expressed sequence is notsubstantially affected. Thus, for the tef promoter and the rRNA clusterpromoter, an “equivalent” thereof retains, substantially, the ability ofthe promoters contained within the sequences to cause expression of geneproduct in a host cell. As discussed above, methods for producing suchequivalents, for example, by PCR-based or oligonucleotide-basedmutational methods or other methods well known in the art. A person ofordinary skill in the art would be able to produce such equivalents withlittle difficulty. Testing for efficacy of the equivalent promoters canbe performed in many ways known to those of average skill in the art.For the tef promoter, promoter function can be determined in E. coli,yeast and Rhizopus species, or another suitable host cell. Similarly,the rRNA cluster promoter can be tested in E. coli, yeast, and Rhizopuscells, or in any other suitable host cell. Expression levels can bedetermined by, for example and without limitation, Northern blot, byquantitative RT-PCR (e.g., TAQMAN) or by expression of an indicator geneproduct.

By the term “vector” is meant a means for introducing a foreignnucleotide sequence into a cell, including without limitation, a plasmidor virus. Such vectors can operate under the control of a host cell'sgene expression machinery. A vector contains sequences that facilitatereplication and/or maintenance of a segment of foreign nucleic acid inthe host cell. Generally, the vector is introduced into a host cell forreplication and/or expression of the segment of foreign DNA or fordelivery of the foreign DNA into the host genome. A typical plasmidvector contains: (i) an origin of replication, so that the vector can bemaintained and/or replicated in a host cell; (ii) a selectable marker,such as an antibiotic resistance gene to facilitate propagation of theplasmid; and (iii) a polylinker site containing several differentrestriction endonuclease recognition and cut sites to facilitate cloningof a foreign DNA sequence. Yep353, discussed below in the Examples, isone such plasmid vector.

RNA interference (RNAi) is a powerful and robust method for disruptinggene expression. It is based on a highly conserved gene silencing methodthat uses double-stranded RNA (dsRNA) or single-stranded RNA (ssRNA,see, e.g., Martinez J, et al., Cell 110(5):563-74 (2002)) as a signal totrigger the degradation of homologous cellular RNA. The mediators of thesequence-specific degradation are 21- to 23-nucleotide (nt) dsRNA smallinterfering RNAs (siRNA). Selection of appropriate siRNA sequences andpreparation of the siRNA are discussed in detail in Elbashir, S. M. etal., Methods 26: 199-213 (2002) and in U.S. Patent Application Nos.2002/0173478, 2002/0182223, 2002/0183276, 2002/0160393 and 2002/0162126.

Xia et al. describes construction of suitable plasmid containing a genefor expression of an siRNA. That reference also describes recombinantviral vectors and delivery systems The reference describes appropriateexpression of an siRNA hairpin which down-regulation of the expressionof a target β-glucuronidase gene in mouse brain and liver, therebyproviding proof of concept of the usefulness of siRNA technology as agene therapy for human diseases (Xia et al., Nature Biotechnology,20:1006-1010 (2002)). See also, for example, U.S. Patent ApplicationNos. 2004/0241854 and 2004/0053876. Vectors for siRNA production arewidely available from commercial sources, such as, without limitation,Ambion, Inc. of Austin Tex., Invivogen of San Diego, Calif., andGenScript Corporation of Piscataway, N.J. Vectors containing appropriatepromoters, such as Pol III promoters, include for example and withoutlimitation, H1 and U6 promoters and have proven especially useful inproducing sufficient quantities of siRNA. A typical siRNA “gene” wouldtherefore comprise an appropriate promoter operably linked to a sequenceencoding an siRNA. Ambion's Technical Bulletin #506 (“siRNA DesignGuidelines”) provides non-limiting examples of siRNA designconsiderations. Computer software for generating suitable siRNAsequences from, for example and without limitation, a cDNA or ORFsequence also is commercially available.

Using well-established methods for determining effective siRNAsequences, siRNA sequences can be made to silence R. oryzae pyruvatedehydrogenase and pyruvate decarboxylase. One non-limiting example of ansiRNA sequence designed to silence the pyruvate dehydrogenase sequencefrom R. oryzae (FIG. 5) is:

Sense 5′-CAGACGAUGACCUUCCUUA (SEQ ID NO:12) Antisense5′-UAAGGAAGGUCAUCGUCUG (SEQ ID NO:13)

One non-limiting example of an siRNA sequence designed to silencepyruvate decarboxylase from Rhizopus oryzae (GenBank Accession Nos.AF282846 and AF282847) is:

Sense 5′-CUUUGAUGUGUUCUUCAAC (SEQ ID NO:14) Antisense5′-GUUGAAGAACACAUCAAAG (SEQ ID NO:15)

In one example, the sense/antisense pairs provided above may beexpressed under the control of the P_(TEF) promoter or rRNA clusterpromoter in a vector construct, such as for example and withoutlimitation in pPYR225b containing the pyrG gene for selection.

Along with RNAi, antisense RNA is another method of interference withgene function. In antisense technology, RNA complementary to portions ofmRNA are introduced into a cell, thereby down-regulating production ofthe protein product of the mRNA. Unlike RNAi technology, antisensensedoes not completely silence the target gene in most cases. Production ofuseful antisense constructs and reagents are well within the abilitiesof those of ordinary skill in the art. At least 450 U.S. patentsdirected to antisense technologies and applications thereof have beenissued to date.

In one example, U.S. Pat. No. 6,838,283 describes antisense modulationof survivin, which is accomplished by providing antisense compoundswhich specifically hybridize with survivin mRNA. As described in thatpatent, the specific hybridization of an antisense sequence with itstarget nucleic acid (“target nucleic acid” encompasses DNA encoding thegene to be modulated), as well as RNA (including pre-mRNA and mRNA)interferes with the normal function of the nucleic acid. The functionsof DNA to be interfered with include replication and transcription. Thefunctions of RNA to be interfered with include, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in or facilitatedby the RNA. The overall effect of such interference with target nucleicacid function is modulation of the expression of the gene to bemodulated. “Modulation” therefore means either an increase or a decreasein the expression of a gene or its product.

In some embodiments, the target is a nucleic acid molecule encodes, forexample, pyruvate dehydrogenase, wherein expression of the moleculeshunts pyruvate towards the production of lactate, ethanol and/orfumarate and away from the mitochondrial Krebs cycle. In yet otherembodiments, the nucleic acid molecule encodes pyruvate decarboxylase,thereby shunting pyruvate away from ethanol production. Down-regulationof both pyruvate dehydrogenase and pyruvate decarboxylase favorsproduction of fumarate. It is necessary to determine a site or siteswithin a gene for the antisense interaction to occur such that thedesired inhibition of gene expression will result. Within the context ofthe present invention, an intragenic target for the antisense compoundcan be the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the target gene. The ORF can bepyruvate dehydrogenase or pyruvate decarboxylas,e though the ORF of anygiven gene may be used. The translation initiation codon or “startcodon” can be 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule) or any equivalent, for example, genes havinga start codon RNA sequence of 5′-GUG, 5′-UUG, 5′-CUG, 5′-AUA, and 5′ACG. Some genes have two or more alternate start codons, which may alsobe used to initiate translation. As used herein, “start codon” and“translation initiation codon” refer to the codon or codons that areused to initiate translation of an mRNA molecule transcribed from atarget gene, regardless of the sequence(s) of such codons. It is alsoknown in the art that a translation termination codon (or “stop codon”)of a gene may have one of three (RNA) sequences: 5′-UAA, 5′-UAG, and5′-UGA (i.e., the corresponding DNA sequences are 5′-TAA, 5′-TAG, and5′-TGA, respectively).

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively by antisense. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene.Similarly, the 3′ untranslated region (3′UTR) may be targeted, e.g., theportion of an mRNA in the 3′ direction from the translation terminationcodon, including nucleotides between the translation termination codonand 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′cap of a eukaryotic mRNA comprises an N7-methylated guanosine residuejoined to the 5′-most residue of the mRNA via a 5′—5′ triphosphatelinkage. The 5′ cap region of an mRNA is considered to include the 5′cap structure itself, as well as the first 50 nucleotides adjacent tothe cap. The 5′ cap region may also be a preferred target region.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target; that is, theyhybridize sufficiently well and with sufficient specificity, to give thedesired effect. As used herein, “hybridization” means hydrogen bonding,which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogenbonding, between complementary nucleoside or nucleotide bases. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. “Complementarity,” as usedherein, refers to pairing between two nucleotides according to the rulesof nucleotide base-pairing (i.e., A:T/U; C:G). For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position of a DNA or RNAmolecule, then the oligonucleotide and the DNA or RNA are considered tobe complementary to each other at that position. The oligonucleotide andthe DNA or RNA may hybridize to each other when a sufficient number ofcorresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementarity” are terms which are used to indicatea sufficient degree of precise pairing such that stable and specificbinding occurs between the oligonucleotide and the DNA or RNA target. Itis understood in the art that the sequence of an antisense compound neednot be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. An antisense compound is specificallyhybridizable when binding of the compound to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA tocause a down-regulation of the expression of the target DNA or RNA, andthere is a sufficient degree of complementarity to avoid non-specificbinding of the antisense compound to non-target sequences underconditions in which specific binding is desired, that is, underconditions in which the host cell is grown.

A typical antisense construct contains a transcribed portion of the geneto be modulated in antisense orientation. Thus, a typical antisenseconstruct contains a promoter operably linked to a transcribed sequenceor a portion thereof as the expressed sequence and a transcriptionterminator (polyadenylation signal, for example), where the transcribedsequence is oriented in the 3′ to 5′ direction as compared to thewild-type transcribed sequence.

Eukaryotic cells regulate the expression of genes in many ways. Theexpression of many eukaryotic genes, however, is controlled primarily atthe level of transcription. Promoters can specify the time and manner inwhich transcription can occur from a particular gene. Therefore, genescan be effectively regulated by strong promoters. Promoters that drivesuch expression of genes in Rhizopus were heretofore not known.

Two Rhizopus genes described in public databases include the openreading frames of the translation elongation factor (tef) gene (GenBankAccession No. AF157289) and the ribosomal RNA cluster (rRNA cluster)gene (GenBank Accession No. AB109757). These two genes are expressed athigh levels in all eukaryotic cells regardless of growth state or mostenvironmental changes.

The rRNA cluster is a tandem repeat of identical copies of a singlegene. These genes, which encode the precursor of the 18S, 5.8S and 28Sribosomal RNAs, are transcribed in the nucleolus by RNA Polymerase I(“Pol I”). Pol I produces a single primary transcript that is processedpost-transcriptionally to generate all three RNAs. The promoter regionof the rRNA cluster genes spans about 150 base pairs just upstream of(5′ to) the transcription initiation start site. These promoters arerecognized by two transcription factors, upstream binding factor (“UBF”)and promoter selectivity factor-1 (“SL-1”), which bind cooperatively torecruit Pol I to form a transcription initiation complex.

In a particular embodiment, Pol I, along with transcription factors andenhancer elements, binds to the novel promoters of the rRNA clustergenes to regulate expression of the genes. Pol I transcription islocalized to the nucleolus and is not inhibited by α-amanitin, a toxicpeptide found in certain types of mushrooms. Pol I, alone, cannotinitiate or terminate transcription. UBF and SL-1 are necessary andsufficient for full transcription by Pol I. Pol I promoters contain anessential core element immediately surrounding the transcription startsite and an upstream control element (UCE) beginning about 100 basesupstream of the start site. UBF binds both the UCE and an upstreamportion of the core elements.

Pol I termination of transcription occurs at well-defined sites. Thetermination sites, called Sal boxes, specifically terminatetranscription and comprise an 18 base pair consensus sequence. The Salbox serves as the binding site for transcription termination factor I(TTFI). A single Sal box, which is in the proper orientation and towhich TTFI is bound, is sufficient for termination of transcription.

Transcription Elongation Factors (TEFs) are universally conservedproteins that promote the GTP-dependent binding of an aminoacyl-tRNA toribosomal A-site in protein synthesis. Especially conserved is theN-terminus of the protein containing the GTP binding domain. TEFs arevery abundant in cells, comprising about 4-6% of total soluble proteins.Tef genes have been isolated from several organisms. In some organisms,they constitute a multigene family. A number of tef pseudogenes alsohave been isolated from some organisms. Tef is constitutively expressed,except in aging and quiescent cells. Tef is not known to be regulated bythe growth substrates of the host.

Tef promoters are RNA Polymerase II (Pol II) promoters. That is, Pol IIis responsible for transcription of the tef gene. Pol II is responsiblefor synthesizing the precursors to messenger RNA (mRNA) and severalsmall nuclear RNA molecules localizes to the nucleoplasm. Like Pol I,Pol II requires a number of transcription factors to assemble on thepromoter to initiate transcription. One of the best characterized Pol IIpromoter elements is the TATA box. The TATA box consists of a specificsequence of nucleotides (TATAAA) located approximately 25 base pairsupstream of the transcription initiation site. It is present in mosteukaryotic genes that encode mRNA.

The mRNAs transcribed by Pol II are polyadenylated. Polyadenylation issignaled by a poly (A) addition (AAUAAA) (also known as a poly (A) site)at the 3′ end of the processed mRNA. The poly (A) site not onlycontributes to the addition of the poly (A) tail, but also totranscription termination. Transcription is terminated 200 to 2,000bases downstream of the poly (A) site.

In particular embodiments, the present invention is directed to isolatedpolynucleotides that include a promoter from the Rhizopus transcriptionelongation factor (tef) gene and/or from the Rhizopus ribosomal RNAcluster (rRNA cluster) gene. Such sequences may be isolated from anyspecies such as Rhizopus delemar, Rhizopus niveus or Rhizopus oryzae.

In certain embodiments, an isolated polynucleotide may comprise anexpressed sequence, such as an ORF, operably linked to the promoter. Inparticular embodiments, the promoter is operably linked to a proteincoding sequence that encodes an enzyme that increases carbon flux tolactic acid or fumarate production. The increase in carbon flux is aresult of an increase in the transcription of the gene encoding thatenzyme. The protein coding sequence may encode, for example and withoutlimitation, pyruvate carboxylase (e.g., SEQ ID NO:6, SEQ ID NO:7, andSEQ ID NO:8), phosphoenolpyruvate carboxylase (e.g., SEQ ID NO:9),pyruvate dehydrogenase (e.g., SEQ ID NO:4 and SEQ ID NO:5), glucokinase,phosphoglucose isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase,phosphoglycerate kinase, phosphoglycerate mutase, enolase and/orpyruvate kinase. The gene also may encode enzymes that catalyzereactions that regenerate nicotinamide adenine dinucleotide (NAD), forexample, lactate dehydrogenase (ldh).

In other particular embodiments; an isolated polynucleotide comprisesthe nucleotide sequence of base pairs 1-877 or 1-351 of the tef genepromoter of Rhizopus oryzae (FIGS. 2 and 3; SEQ ID NO: 1 and SEQ ID NO:2, respectively). The isolated polynucleotide can also be the nucleotidesequence of base pairs 1-1043 of the rRNA cluster gene promoter ofRhizopus oryzae (FIG. 10; SEQ ID NO: 10; SEQ ID NO: 11) or the core rRNApromoter found therein (SEQ ID NO:11). Alternatively, the isolatedpolynucleotide can also be fused to a reporter gene, for example, butnot limited to, the β-galactosidase (lac-Z) reporter gene fromEscherichia coli.

The invention also includes vectors comprising an isolatedpolynucleotide, wherein the polynucleotide comprises a promoter derivedfrom a Rhizopus tef gene or a Rhizopus rRNA cluster gene. In certainembodiments, the vectors include a multiple cloning site (MCS) 3′ to thepromoter, permitting insertion of an expressed sequence into the vectorto produce the expression product of the expressed sequence, such as aprotein or functional nucleic acid. In yet another embodiment, thevector contains both a promoter derived from a Rhizopus tef gene andfrom a Rhizopus rRNA cluster gene, optionally including an MCS 3′ toboth promoters, permitting insertion of two different expressedsequences in the same vector.

In other embodiments, the vector includes an ORF or coding sequence,with or without introns, for expressing an enzyme that affects carbonflux in a host cell containing the vector. In one embodiment, theexpressed sequence encodes an enzyme, such as ldh, that increases carbonflux to lactic acid production during changes in fermentationconditions. The increase in carbon flux to lactic acid would result froman increase in the transcription of the gene. Any ldh gene can be usedso long as that ldh accepts pyruvic acid as a substrate. For example,any of the genes encoding bacterial ldh described herein can be used. Inone embodiment, the ldh is derived from microorganisms, including butnot limited to, Rhizopus oryzae (GenBank Accession Nos. AF226154 (ldhA)and AF226155 (ldhB)) or Lactobacillus casei (GenBank Accession No.M76708). In yet another embodiment, a host cell comprises a gene inwhich the tef or rRNA cluster promoter is operably linked to a gene thatencodes an enzyme that modifies carbon flux in the host cell, forexample and without limitation, the ldh enzyme or any other suitablegene described above and known in the art.

Likewise, a coding sequence for pyruvate carboxylase (e.g., SEQ ID NO:6,SEQ ID NO:7, and SEQ ID NO:8), phosphoenolpyruvate carboxylase (e.g.,SEQ ID NO:9) would increase flux of carbon from pyruvate orphosphoenolpyruvate, respectively, to oxaloacetate, resulting inincreased production of fumarate through action of malate dehydrogenaseand fumarase (see FIG. 1). Further, carbon flux can be changed bychanging expression of pyruvate dehydrogenase (e.g., SEQ ID NO:4 and SEQID NO:5) in the TCA cycle (see FIG. 1). In yet other embodiments,fumarase (e.g., R. oryzae fumR; GenBank Accession No. X78576) may beoverexpressed to further increase carbon flux to fumarate. In some otherembodiments, the tef and rRNA cluster gene promoters can be fused to aβ-galactosidase lac-Z reporter gene from, for example, Escherichia coli.

Alternatively, carbon flux can be increased by expressing a gene productthat interferes with shunting of pyruvate and its precursors intoundesirable metabolic pathways, for example by interfering with theenzymes involved in the conversion of pyruvate into ethanol, or enzymesinvolved in conversion of 3-phosphoglycerate to glycerol. Genes ofinterest also include pyruvate decarboxylase genes, such as those of R.oryzae (GenBank Accession Nos. AF282846 (pdcA) and AF282847(pdcB)).

In another embodiment, the present invention is directed to a host cellcomprising an isolated polynucleotide, wherein the polynucleotidecomprises a promoter derived from a Rhizopus tef gene promoter or aRhizopus rRNA cluster gene promoter. Microorganisms capable of acting asa host cell include, but are not limited to, Representative microbialhost cells include, but are not limited to, fungal cells such asRhizopus ssp., Saccharomyces ssp., Streptomyces ssp., Pichia ssp.,Aspergillus ssp., and bacterial cells such as Lactobacillus ssp.,Escherichia ssp., Corynebacterium ssp., Brevibacterium ssp., Pseudomonasssp., Proteus ssp., Enterobacter ssp., Citrobacter ssp., Erwinia ssp.,Xanthomonas ssp., Flavobacterium ssp., Streptococcus ssp., Lactococcusssp., Leuconostoc ssp., and Enterococcus ssp.

Nucleic acids can be introduced into cells according to standardmethodologies including electroporation, or any other transformation ornucleic acid transfer method known in the art. For example, R. oryzaecan be transfected by electroporation. R. oryzae cells can bepermanently transformed by insertion of a gene of interest into the cellby electroporation, so long as the introduced DNA integrates into thehost cell genome. This is accomplished, without any intention to bebound by this theory, by homologous recombination of the introduced DNAwith the genomic DNA via single or double crossover, or is randomlyintegrated. The efficiency of transformation is increased when theintroduced DNA is linearized and contains non-complementary ends, as isthe case when a DNA fragment containing a gene is excised from a plasmidusing two different restriction endonucleases which yieldnon-complementary ends. In such instances, the sequence can be purifiedfrom the plasmid backbone prior to transfection. Circularized DNA tendsto concatamerize in R. oryzae, yielding large, circular extrachromosomalelements, which are eventually lost from the host cell during successivepassage of the transfected cell line. Linearized DNA havingcomplementary ends can also re-circularize and concatamerize (notnecessarily in that order) and be lost in the same manner as anextrachromosomal element during successive passage of the transfectedhost cell line.

Host cells may be cultured under any conditions, such as those known inthe art. As stated previously, fermentation conditions can affect theflux of carbon in an organism. For example, strong aeration shifts theflux of carbon to production of acetic acid and acetoin, and away fromlactic acid production in lactic acid-producing bacteria. Fermentationconditions include, without limitation: the level of aeration, pH, andoxygen saturation level of the medium, as well as the amount of carbonand other growth factors available in the medium. The carbon source canbe, for example and without limitation, various sugar alcohols, polyols,aldol sugars or keto sugars, including but not limited to arabinose,cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose,mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose,trehalose, pyruvate, succinate or methylamine or other substrates whichmay be determined by one skilled in the art. As described herein, manyorganisms will thrive on common growth media. For example and withoutlimitation, R. oryzae can be grown in LB (Luria-Bertani) Broth.

Host cells may also be engineered to change carbon flux. Provided in oneembodiment is a method of increasing carbon flux to lactic acidcomprising culturing in a culture medium a host cell comprising aRhizopus tef gene promoter, a Rhizopus rRNA cluster gene promoter, oranother promoter operably linked to an ldh coding sequence andrecovering lactic acid from the culture medium. Likewise, a method ofincreasing carbon flux to fumarate is provided comprising culturing in aculture medium a host cell comprising a Rhizopus tef gene promoter, aRhizopus rRNA cluster gene promoter, or another promoter operably linkedto a pyruvate carboxylase or phosphoenolpyruvate carboxylase codingsequence and recovering fumarate from the culture medium.

In another embodiment, regulation of the expression of a gene productincludes providing a coding region that encodes a gene product; operablylinking the coding region to an isolated tef gene promoter or an rRNAcluster gene promoter to form a promoter-coding region within genomicDNA in cells wherein the promoter regulates the expression of the geneproduct in the cells. In some embodiments, the promoter-coding regioncan be integrated into a genomic DNA in cells wherein the promoterregulates the expression of the gene product in the cells.

In yet another embodiment, the methods of manipulating carbon flux in acell, such as, without limitation a R. oryzae cell, are provided.Referring to FIG. 1, expression of a number of genes may be utilized toengineer a cell with altered metabolic pathways. As discussed in detailabove, the expression of certain genes native to the host cell, forexample and without limitation, R. oryzae, such as ldh (to producelactate), pyruvate carboxylase (to produce oxaloacetate), fumarase (forexample and without limitation, R. oryzae fumR, GenBank Accession No.X78576; and Freidberg, et al., Gene. 163(1):139-44(1995)) (to producefumarate), or, glycerol-3-phosphate dehydrogenase (to produce glycerol)can be increased by the methods described herein. In a similar manner,genes not native to the host cell may be introduced into the host cellunder constitutive or inducible control of a promoter with the goal ofincreasing carbon flux to a desired end-product metabolite, such asfumarate. In one example for production of fumarate, alfalfa (Medicagosativa) phosphoenolpyruvate (PEP) carboxylase is introduced into an R.oryzae cell to shunt carbon from PEP directly to oxaloacetate,preventing diversion of pyruvate to the Krebs cycle and to ethanol andlactate production (see FIG. 1, dotted line).

In a further example, antisense or RNAi technologies may be used alone,or in combination with increased gene expression of lactatedehydrogenase, PEP carboxylase or pyruvate carboxylase to further divertcarbon from one metabolic pathway to another. It is noted that undersome conditions, complete gene silencing may prevent sufficient cellculture growth unless a specific metabolite is provided in the culturemedium (auxotroph). Thus, production of a bradytroph may be optimized inmany instances with antisense technology or RNAi technology. Oneparticular candidate enzyme for antisense or RNAi targeting is pyruvatedehydrogenase, which converts pyruvate to acetyl coenzyme A(acetyl-coA), which donates its acetyl group to oxaloacetate to formcitrate in the citric acid cycle, resulting in overproduction offumarate. A cell co-transfected with genes for overexpressing pyruvatecarboxylase and for down-regulating expression of pyruvate dehydrogenaseis expected to shunt carbon to fumarate. Use of an inducible promoter,such as the TET-ON or TET-OFF promoter (BD Biosciences Clontech) canavoid the growth inhibition connected with the silencing of pyruvatedehydrogenase. In such a case, the cells can be grown to a desireddensity in culture before pyruvate dehydrogenase (and the Krebs cycle)is silenced.

Another embodiment includes a method to construct selectable markers formore efficient metabolic engineering of a microorganism, comprisingintroducing into a lactose auxotroph host cell a nucleic acid comprisinglac-Z (encoding β-galactosidase) operably linked to a promoter derivedfrom a Rhizopus tef gene or a Rhizopus rRNA cluster gene. The nucleicacid can be a vector containing a second gene for expression in the hostcell. The ability of a transfected host cell to grow on lactose wouldfacilitate selection of transfected host cells.

In yet an additional embodiment, the compositions of the invention maybe produced at a first geographic location and transported or shipped toa second geographic location. For instance, a facility at the firstgeographic location may be able to produce a product more economicallythan a facility at the second location due to various factors. Thefactors may include, inter alia, lower costs of materials (i.e., themannitol), lower costs of energy (i.e., electricity or gas), lower costsof labor (i.e., wages paid to employees), lower costs of environmentalcontrols or effects, or any other requirement for production of thecompositions of the invention. Further, a certain product may be wellsuited for production in the first geographic location and desired, butnot produced well, in the second geographic location. As a non-limitingexample, residents of Alaska may desire bananas produced in CentralAmerica. Thus, the costs of producing the products in a first geographiclocation may be less than the costs of producing the products in asecond geographic location, resulting in the production costs of theproduct being less in the first geographic location.

In such an instance, the compositions of the invention may be producedat the first geographic location and shipped to the second geographiclocation, such as by transport over water with ships or barges,trucking, flying, or other means of transportation. The geographiclocation may be a county, a state, a country, a continent and/orcombinations of any thereof. In this manner, the product may be producedin a first country and transported and/or sold in a second country.

The following are examples of methods and compositions of the invention.The examples are not meant to limit the scope of the invention, asdefined by the claims.

EXAMPLE 1 Isolation of the tef Gene Promoters and Rhizopus ETS Region

Promoter regions of the tef and regions of the External TranscribedSequences (ETS) were cloned by cutting total genomic DNA of R. oryzaewith restriction endonuclease. The DNA was ligated to adapters (LA PCRin vitro Cloning Kit, Takara Mirus Bio, Inc. of Madison, Wis., see alsoU.S. Pat. No. 5,436,149) and the promoter regions were amplified withthe polymerase chain reaction using one primer complementary to knowngene sequences and one primer complementary to the adapter, as follows:

(SEQ ED NO:16) C2 cassette primer 5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAG;(Takara) (SEQ ID NO:17) TEF reverse primer5′-GTAATCATGTTCTTGATGAAATCACGG; (SEQ ID NO:18) ETS reverse primer5′-GATTCACTGAATATGCAATTCACACTAG.

Three products were amplified using the respective primers. Theresulting products were a 351 base pair tef polynucleotide (FIG. 2), a877 base pair tef polynucleotide (FIG. 3) and an ETS polynucleotide(FIG. 4). The 351 base pair tef polynucleotide (FIG. 2) was theninserted into the multiple cloning site to the E. coli β-galactosidaselac-Z reporter gene of YEP353 plasmid (GenBank Accession No. U03500).

Yep353 (GenBank Accession No. U03500) is a shuttle vector that hasorigins of replication for bacteria and yeast. It has a multiple cloningsite situated in front of the reporter gene lacZ. When a piece of DNAthat responds to transcriptional machinery is cloned into it in theproper direction, the lacZ gene is expressed and β-galactosidaseactivity is quantifiable. If nothing is cloned in the MCS, or if theinsert DNA in the MCS does not behave as a promoter in these organisms,then no activity is expressed.

The 351 base pair tef polynucleotide showed strong expression in E. coliand yeast. E. coli cultures containing the YEP353 PTEF:lacZ plasmid weregrown on LB plates containing X-gal. The strong promoter capability oftef in E. coli and yeast indicates that it has a broad host celltropism, making the promoter useful in a large variety of organisms.

EXAMPLE 2 Effect of Promoter Constructs on Lactic Acid Production

In another construct, the 351 base pair tef polynucleotide is fused toldh genes from Rhizopus oryzae and Lactobacillus casei. The effect ofover-expression of these genes on lactic acid production can beevaluated.

EXAMPLE 3 Reporter-Promoter Constructs

In another construct, the 351 base pair tef polynucleotide was fused tothe β-galactosidase lac-Z reporter gene from Escherichia coli. Thisconstruct can be used to select for transformants that show a gain ofability to use lactose as a carbon source and can facilitate moreeffective metabolic engineering. With this construct, an auxotroph willnot need to be created before genetic engineering begins.

EXAMPLE 4 Construction of P_(TEF):pyrC:T_(PGK) Plasmid—Using LigationIndependent Cloning

TEF promoter (PTEF) is amplified by PCR using primers that will create asmaI site on the 5′ end and add the ATG codon and 10 bases of the 5′ endof pyruvate carboxylase to the 3′ end of the amplicon (Product1=SmaI-PTEF-ATG-10 bp of pyrC). Pyruvate carboxylase (pyrC) from cDNAclone is amplified by PCR using primers that will add 10 base pairs ofthe 3′ end of PTEF and the ATG codon to the 5′ end of the amplicon andan speI restriction site to the 3′ end of the amplicon (Product 2=10 bpof PTEF-ATG-pyrC-SpeI). PCR products 1 and 2 are mixed with P_(TEF) smaIforward primer and pyrC spel reverse primer. P_(TEF):pyrC product isamplified by PCR. The smaI-PTEF:pyrC-speI, PCR product 3, and pyr225bvector are cut with small and speI and ligated. S. cerevisiae PGKterminator (T_(PGK), terminator on vector YIpDCE1 GenBank-AF039102) isamplified by using primers that will introduce speI and sacI restrictionsites. Pyr225b containing P_(TEF):pyrC and the amplified Tpgk terminatorare cut with speI and sacI and ligated. pPYR225B vector (pBluescriptKS-containing a 2.25 Eco RI genomic Rhizopus fragment (GenBank AF497632)contains the pyrG gene.

The resulting plasmid is linearized within the pyrG gene to facilitateType I or single crossover into genomic DNA, and used to transform apyrG deletion mutant generated from Rhizopus oryzae wild-type strainATCC 10260 (NRRL 1526).

EXAMPLE 6 Construction of P_(TEF):pepc:T_(PGK) Plasmid—Using LigationIndependent Cloning

The plasmid construction outlined in Example 5 above can be used tobuild a similar construct containing the alfalfa PEP carboxylase codingsequence (see U.S. Pat. No. 6,599,732, SEQ ID NO: 1). The pyrC fragmentof P_(TEF):pyrC:T_(PGK) can be excised with restriction endonucleasessbfI and apaI and replaced with a PEP carboxylase ORF that has beenPCR-modified to contain sbfI and apaI restriction sites.

EXAMPLE 7 Rhizopus oryzae Pyruvate Carboxylase

The isolation and characterization of Rhizopus oryzae genomic and cDNAis described herein. Both the nucleic acid molecule and the encodedpyruvate carboxylase protein are provided. The properties of this enzymeand potential application for fumaric acid and during lactic acidproduction are discussed.

As part of an effort to characterize the genes encoding the enzymes inthe pathway leading to the synthesis of lactic acid, fumaric acid,ethanol and glycerol during fermentation, a pyruvate carboxylase genewas isolated from R. oryzae and the relatedness of its deduced proteinto other known orthologs was studied. Two degenerate oligonucleotideprimers were synthesized based on conserved regions pyruvatecarboxylase-related amino acid sequences of A. bisporus (GenBankAccession No.: AJ276430), A. terreus (GenBank Accession No.: AF097728),P. pastoris (GenBank Accession No.: Y11106), and S. pombe (GenBankAccession No.: D78170,). Amplification by polymerase chain reaction(PCR) with R. oryzae genomic DNA as template yielded a product of thepredicted size (648 bp). Additional PCR reactions using gene-specificand degenerate primers were used to isolate the pyruvate carboxylasegene and cDNA fragments from R. oryzae. The cDNA, genomic DNA, andencoded amino acid sequence of the protein, were described (SEQ IDNOS:6, 7, and 8, respectively). The R. oryzae gene (designated PYC) hasan intron of 61 bp. The 3′-untranslated region is 91 bp and the5′-untranslated region is 168 bp in length (FIG. 6).

Rhizopus oryzae strain 28.51 was maintained on YM agar plates (perliter:3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g dextrose,and 20 g agar). The fungus was grown in YML liquid media (per liter: 3 gyeast extract, 3 g malt extract, 5 g peptone, and 10 g dextrose) at roomtemperature with shaking (100 to 150 rpm) or YM agar plates at 30° C.

DNA and total RNA were extracted from frozen spores (−80° C.) of R.oryzae. Genomic DNA was isolated using the Omniprep™ purification system(Geno Technology, Inc., St. Louis, Mo.) or by a CTAB buffer (100 mMTris-HCl, pH 7.5, 1% mixed alkyltri-methyl ammonium bromide (Sigma, St.Louis, Mo.), 0.7M NaCl, 10 mM EDTA 1% β-mercaptoethanol (v/v)) plus0.03% proteinase K. The frozen spores were ground by mortar and pestleand extracted in the CTAB buffer followed by incubation at 65° C. for 30min. One volume of chloroform/isoamyl alcohol (24:1) was added, gentlymixed for 5 min., and centrifuged at 3,000 rpm for 20 min. Thesupematant was removed and a ⅔ volume of 2-propanol was added andrecentrifuged as above. The precipitated DNA was rinsed with 75% ethanoland suspended in 0.5 ml sterile water. Contaminating RNA was removed byaddition of 5 μl of 10 mg/ml RNAse A and incubation at 37° C. for about30 min.

Total RNA was isolated using RNAqueous™ Kit (Ambion, Inc., Austin, Tex.)and mRNA was purified from the total RNA using the PolyATtract™ mRNAIsolation Systems (Promega Corporation, Madison, Wis.). The methods usedfor DNA and RNA electrophoresis have been described elsewhere (Sambrook,J., Fritsch, E. F., and Maniatis, T., in Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989)).

PCR was performed in a GeneAmp PCR System 9700™ (Applied Biosystems,Foster City, Calif.) using Taq DNA polymerase (Life Technologies,Gaithersburg, Md.) and two degenerate primers based on conserved aminoacid sequences of pyruvate carboxylase homologs from Aspergillusagricarus, A. terreus, Pichia pastoris, and Schizosaccharomyces pombe.Forward primer 5′CARAGRAGRCAYCARAARGT 3′ (SEQ ID NO:19) is based on theamino acid sequence “QRRHQKV,” and reverse primer 5′TCRTCDATRAANGTNGTCCA 3′ (SEQ ID NO:20) is based on the amino acidsequence “WTTFIDD” (where Y=T or C; R=G or A; D=A, G or T; N=A, T, G, orC) (SEQ ID NO:21). The degenerate primers were used in Touchdown PCR(Don, R. H., et al., Nucleic Acids Res. 19:4008 (1991)) to amplify a648-bp fragment from R. oryzae genomic DNA. Touchdown PCR was performedunder the following conditions: initial denaturation at 94° C. for 3min; 38 cycles of denaturation, 94° C. for 30 sec; annealing for 30 sec;and polymerization at 72° C. for 2 min. The annealing temperature rangedfrom 55° C. to 45° C. with a decrease of 1° C. every three cycles. Thiswas followed by 14 cycles of denaturation at 94° C. for 1 min; annealingat 45° C. for 30 sec.; and polymerization at 72° C. for 2 min. The PCRproduct was cloned into pGEM T-easy™ vector (Promega, Madison, Wis.).Additional PCR products were isolated using pyruvate carboxylase (PYC)gene-specific primers, genomic DNA or cDNA and other degenerate primers.

The 5′ end of the pyruvate carboxylase (PYC) cDNA was determined usingthe GeneRacer™ kit, following the instructions of the manufacturer(Invitrogen Corporation, Carlsbad, Calif.). A PYC-specificoligonucleotide of sequence 5′-CCAATACGACCGAGTTGATAGGATTCAT-3′ (SEQ IDNO:22) was used to prime first-strand cDNA synthesis, which was thenamplified by PCR using a nested primer of the sequence5′-GCATAGATAATGTATCTTCATGA-3′ (SEQ ID NO:23).

Automated fluorescence DNA sequencing was done at the W. M. Keck Centerfor Comparative and Functional Genomics Facility, University of Illinoisat Urbana-Champaign. Sequence data were analyzed with DNASTAR™ software(DNASTAR, Inc., Madison, Wis.).

The open reading frame of the product of PYC, PYCp, is 1178 amino acidsand has a molecular mass of 130 kD. PYCp has ˜61 to 67% overall identitywith S. cerevisiae (Morris, C. P., et al., Biochem. Biophys. Res.Commun. 145:390-396 (1987)); Aspergillus niger (Panneman, H., Ruijter,G. J. G., Van den Broeck, H. C. and Visser, J., unpublished); A. terreus(Li, Y. F., Chen, M. C., Lin, Y. H., Hsu, C. C. and Tsai, Y. C.,unpublished); P. pastoris (Menendez, J., et al., Yeast 14:647-654(1998)); and S. pombe (Saito, A., et al., unpublished) pyruvatecarboxylase proteins. The similarity is very strong throughout theprotein sequence (FIG. 8). The two ATP and biotin binding domains are100% conserved, while the pyruvate binding domain is 89% conserved amongthese fungal proteins (FIG. 8), like its yeast homolog (Lim, F., et al.,Arch. Biochem. Biophys. 258:259-264 (1987)). The PSORT program (Nakai,K., et al., Genomics 14:897-911 (1992)) strongly predicts thesubcellular localization of R. oryzae pyruvate carboxylase to thecytoplasm. The computed probability of PYCp having a cytoplasmiclocalization is 78%. Hybridization of a PYC probe to a blot of R. oryzaegenomic DNA digested with different restriction enzymes (PstI, BamHI, orEcoRI) resulted in a single band in one case and multiple bands in theothers. Preliminary data indicates that there may be a single copy ofthis pyruvate carboxylase gene in R. oryzae (FIG. 9).

The production of fumaric acid by R. oryzae has been shown to resultfrom a cytosolic pathway during which pyruvate is converted tooxaloacetate by pyruvate carboxylase (Osmani and Scrutton, Ann NY AcadSci 447: 56-71 (1985)). Therefore, this gene expression can be enhancedby introducing multiple copies or expressing it from a strong promoterto increase fumaric acid production. Moreover, the disruption of thisgene can also lead to the reduction of fumaric acid produced duringlactic acid production by R. oryzae.

EXAMPLE 8 Cloning and Construction of P_(rRNA cluster) Plasmid

A search of GenBank for the 28S-IGS-18S region for yielded a Rhizopusoligosporus sequence (GenBank Accession No. AY847625). A cloned aportion of the 18S gene upstream from the GenBank sequence ab109757 wasused for the search. Two putative rRNA clusters were found (AY847625 andand other fungal 5S sequences). These were aligned with the two putativerRNA clusters to identify the promoter region of interest. Thus, byusing GenBank sequence AY847625 from R. oligosporus to blast the publicbut un-annotated genome sequence for R. oryzae (Broad Institute), theputative desirable 18S promoter sequence of NTS2 was identified. Primerswere designed against this sequence and a fragment isolated. The forwardprimer was (EcoRi restriction site in italics):

TCGAATTCGGGGGACCACATGGGAATAC (SEQ ID NO:24)The reverse primer was (PstI restriction site in italics):

TGGCTGCAGGTCATGTTGGCAGGATC (SEQ ID NO:25)Using the methods described in Example 1, the isolated fragment (SEQ IDNO:10) was operably linked in frame to the lacZ expression marker andcloned into a Yep353 plasmid. Expression of lacZ was detected in E.coli, but not yeast. This is as predicted since in eukaryotes the rRNAcore promoter (e.g., SEQ ID NO:11) recruits polymerase I (Pol I) and PolI transcripts do not have a 7-methylguanylate cap nor are they recruitedto ribosomes for translation. Conversely, in prokaryotes, transcriptionand translation are simultaneous and capping is not present sotranslation of the protein occurs. Thus, the rRNA promoter can be usedas a promoter in eukaryotes for transcription of antisense or RNAiconstructs.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or anyembodiment thereof. It will be appreciated by those skilled in the artthat changes could be made to the embodiments described herein withoutdeparting from the broad concept of the invention. It is understood,therefore, that this invention is not limited to the particularembodiments disclosed, but is intended to cover modifications that arewithin the spirit and scope of the invention as defined by the claims.

1. An isolated or recombinant nucleic acid molecule comprising an expressed sequence operably linked to SEQ ID NO:2.
 2. The isolated or recombinant nucleic acid molecule of claim 1, wherein the expressed sequence encodes an mRNA.
 3. The isolated or recombinant nucleic acid molecule of claim 2, wherein the mRNA encodes a protein selected from the group consisting of lactate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase.
 4. The isolated or recombinant nucleic acid molecule of claim 1, wherein the expressed sequence encodes an siRNA or an antisense RNA.
 5. The isolated or recombinant nucleic acid molecule of claim 2, wherein the isolated or recombinant nucleic acid molecule is fused to a gene that encodes an enzyme that increases carbon flux to fumarate.
 6. The isolated or recombinant nucleic acid molecule of claim 2, wherein the isolated or recombinant nucleic acid molecule is fused to a gene that encodes an enzyme that increases carbon flux to lactic acid.
 7. A vector comprising: SEQ ID NO:2; and a coding region operably linked to SEQ ID NO:2.
 8. The vector of claim 7, wherein the coding region is selected from the group consisting of an open reading frame, a sequence encoding an antisense RNA, and a sequence encoding an interfering RNA.
 9. The vector of claim 7, wherein the coding region comprises a nucleic acid molecule encoding a protein selected from the group consisting of lactate dehydrogenase, pyruvate carboxylase and phosphoenolpyruvate carboxylase.
 10. An isolated recombinant host cell comprising the vector of claim
 7. 11. The isolated recombinant host cell of claim 10, wherein the isolated recombinant host cell is of a genus selected from the group consisting of Rhizopus, Saccharomyces, Streptomyces, Pichia, Aspergillus, Lactobacillus, Escherichia coli, Corynebacterium, Brevibacterium, Pseudomonas, Proteus, Enterobacter, Citrobacter, Erwinia, Xanthomonas, Flavobacterium, Streptococcus, Lactococcus, Leuconostoc, and Enterococcus. 